Project Summary: Lipid Biomarker Efflux from the Brain following TBI, LaPlaca, M.C., Fernndez, F.M. Traumatic brain injury (TBI) is a major health problem in the US and worldwide, affecting at least 2.5 million Americans every year. TBI is extremely variable from person to person, making the standardization of injury classification and diagnosis challenging. Biomarkers can potentially provide individualized diagnostic information, yet none are currently approved for clinical use. The objective of the proposed work is to identify novel lipid biomarkers for TBI diagnosis and the routes by which they exit the brain as a function of time and injury severity. The overall hypothesis is that TBI-specific lipids will pass both through the blood brain barrier (BBB) and glymphatic system to the blood post-TBI, and that efflux of biomarkers through different routes will have a variety of post-injury temporal patterns. Lipid biomarkers are promising for several reasons: lipids are abundant in brain, are extremely vulnerable to inflammatory and free radical attack, and may leak out of the brain more readily than proteins, rendering them ideal candidates for peripheral diagnostics. Three mutually- informing aims will be pursued to test the following hypotheses: 1) The absolute magnitude of lipid alterations in brain, blood, cerebrospinal fluid, and lymph will increase with injury severity; 2) More than one route of brain clearance of TBI-generated lipid biomarkers exists and the efflux dynamics depend on lipid size and time post- injury; and 3) Efflux transporters and glymph drainage contribute to post-TBI lipid biomarker efflux in addition to diffusion across brain-to-blood barriers. Preliminary discovery metabolomics data identifying lipids in serum that successfully discriminates between injured and uninjured rats will be expanded to include surveillance in cerebrospinal fluid and lymph out to four weeks post-TBI for mild and moderate TBI in both male and female rats. In addition, different clearance paths will be examined, as efflux routes may change with evolving secondary injury pathology. The route for biomarker clearance from the brain to the blood has been assumed to be primarily via the BBB, however recent identification of dural lymph vessels and a glymphatic route for protein biomarker release after TBI changes the focus from BBB-only transport to one of potentially multiple efflux routes. Given the promise of TBI-specific lipid biomarkers and the unknown dynamics of lipid biomarker release, there is a need to understand lipid clearance routes following TBI. Through this research, we expect to identify novel lipid biomarker panels and determine the major route(s) for their release from the brain. This is significant and novel because, while biomarkers provide a unique window into secondary injury events, changes in efflux patterns directly impact clinical interpretation and implementation.